Liquid chromatography systems are used to carry out chemical separations. A typical liquid chromatography system consists of the following major components: a pump, an injector, a column, and a detector. The pump compels a mobile phase, for example, a solution, through a fluid path comprising an injector, column and a detector. The injector permits the introduction of samples into the fluid stream above the column. The column contains a packed bed of media. The media is normally porous and relatively inert. Compounds in the sample will exhibit a characteristic affinity to the media. That is, some compounds exhibit high affinity and some compounds exhibit low affinity. As a result, as the compounds are carried through the media, the compounds separate into bands which elute or come off the column at different times. These bands are detected by the detector.
Absorbance detectors are one exemplary type of detector that can be used to detect the bands eluting from the column. Broad spectrum or bandwidth limited light is directed through a sample, and then measured at the chosen analytical wavelengths by a detector, such as a photodetector. In these instruments, light traverses a fixed distance (a path length) through the sample. The instrument's photodetector signal is measured when the analyte sample concentration is zero (I0) and when the analyte is present (I). Absorbance (A), a dimensionless number commonly expressed in absorbance units (AU) for convenience, is calculated from log(I0/I) and displayed as the instrument output. Absorbance is proportional to the product of path length (b) and concentration (c). This relationship between absorbance, path length, and concentration is known as Beer's Law. A constant of proportionality can be found from a calibration experiment using known analyte concentrations, thus enabling unknown concentrations to be measured. If path length is expressed in centimeters (cm) and concentration in moles per liter (moles/L), then the proportionality constant is called the molar absorbtivity (ε) with units cm−1 (moles/L)−1.
Since the molar absorbtivity, ε, varies with wavelength for any analyte, the instrument can include a monochromator, filters, a diode array spectrograph or, in the case of the infrared, a Fourier transform interferometer, to measure absorbance at specific wavelengths.
The range of analyte concentrations that can be measured by an absorbance detector is limited. At the low end, the minimum detectable change in absorbance is set by the base line noise on the absorbance output, a value which varies from wavelength to wavelength and from instrument to instrument. For example, a well-designed UV absorbance detector for HPLC can detect an absorbance change in the range of about 10μ AU to about 20μ AU. An upper limit of concentration measurement is reached when the relationship between absorbance and concentration becomes significantly nonlinear. This typically occurs when absorbance exceeds about 1 to about 2 AU. The upper absorbance limit is usually the result of stray light or inadequate spectral resolution. The upper absorbance limit varies with wavelength and from instrument to instrument, and is reduced if the solvent or HPLC mobile phase absorbs. The analyte concentration range can be defined as the ratio of the maximum to minimum concentration. Due to the limitations discussed above, the dynamic range of a typical detector is limited to about five orders of magnitude.
A wide dynamic range detector is necessary when very small and very large peaks need to be quantitated in the same chromatogram. For example, the related substances assay commonly performed for the analysis of impurities and degradants in pharmaceutical substances relies on the ability of the absorbance detector to provide sufficient dynamic range to capture both the impurities (concentrations≤0.1%) and the active ingredients (concentrations nominally 100%).
Assays are commonly developed to provide a peak height for the principal component, e.g., an Active Pharmaceutical Ingredient (API), within an acceptable error range with respect to an ideal linear calibration curve. Absorbance detectors are commonly characterized by a linearity specification based on ASTM E685-79 which defines a protocol to determine the absorbance at which the deviation from linearity is five percent.
Absorbance detector design is close to limits imposed by the physics of available components (light sources, photodetectors etc.), the constraints on cell volume required to maintain chromatographic resolution, and market-driven requirements of spectral range and resolution. Currently available absorbance detectors exhibit noise that approaches the shot noise limit of the semiconductors used as photodetectors. Further reduction of the noise will require more intense light sources and/or cooling of the photodiodes to reduce the shot noise limit. As a result, significant improvement of the noise limit through detector design is unlikely and would result in increased cost and/or complexity.
Long path length light-guiding flow cells offer a way to increase concentration sensitivity for a given baseline noise. Unfortunately, the high concentration limit, set by the detector's linear absorbance range, is reduced by the same amount, so that the concentration range remains the same. Moreover, if the mobile phase absorbs, the concentration range will actually be less with a longer cell.
Accordingly, there remains a need for absorbance detectors and associated methods that provide a wide dynamic range.